A subset of humans infected with HIV-1 develops broadly neutralizing antibody responses to multiple epitopes on the envelope glycoproteins (Env), including the CD4-binding site (CD4bs) and VI/V2 region. However, immunization with Env-based vaccine candidates has yet to elicit such effective responses for reasons that are not yet fully understood. To help understand and bypass this roadblock, we have developed new bioinformatics methods for analysis of B cell deep sequencing data to provide a high-resolution view of the antibodyome and its response to antigenic stimuli. These new tools provide an opportunity for significant advances in understanding the humoral immune response to HIV-1 Env immunogens. The goals of our Project are in two distinct areas. First, we will analyze deep sequencing data of B cell transcripts, obtained from the NHP immunization experiments described in detail in Projects 1 and 2 (and performed in Core B) to characterize the antibody populations that arise in response to immunization and their development pathways. Our second focus will be to use x-ray crystallography to determine atomic-level structures of high-interest antibodies we identify, and their relevant antibody/antigen complexes. All of these analytical efforts will be evaluated and synthesized with data from Projects 2 and 3 to directly feedback to Project 1 to inform Env immunogen redesign to impact elicited B cell responses in the second round of planned NHP experiments in Year 4 of this proposal. Our Specific Aims are as follows: (1) To use deep sequencing of memory B cell transcripts to identify and genetically characterize NHP antibodies elicited by soluble Env trimers. These studies will identify the the V, D, and J gene segments used, and otherwise characterize the antibody response. In the second round of NHP experiments, we will use new immunogens optimized by molecular design or in vitro evolution, described in Project 1. (2) We will define the maturation pathways for select identified antibodies; and (3) We will determine structures of elicited antibodies of high interest, and relevant antibody/antigen complexes.